Cell shape regulates subcellular organelle location to control early Ca2+ signal dynamics in vascular smooth muscle cells.

The shape of the cell is connected to its function; however, we do not fully understand underlying mechanisms by which global shape regulates a cell's functional capabilities. Using theory, experiments and simulation, we investigated how physiologically relevant cell shape changes affect subcellular organization, and consequently intracellular signaling, to control information flow needed for phenotypic function. Vascular smooth muscle cells going from a proliferative and motile circular shape to a contractile fusiform shape show changes in the location of the sarcoplasmic reticulum, inter-organelle distances, and differential distribution of receptors in the plasma membrane. These factors together lead to the modulation of signals transduced by the M3 muscarinic receptor/Gq/PLCß pathway at the plasma membrane, amplifying Ca2+ dynamics in the cytoplasm, and the nucleus resulting in phenotypic changes, as determined by increased activity of myosin light chain kinase in the cytoplasm and enhanced nuclear localization of the transcription factor NFAT. Taken together, our observations show a systems level phenomenon whereby global cell shape affects subcellular organization to modulate signaling that enables phenotypic changes.

Resolution of a signal transfer region from a general binding domain in gbeta for stimulation of phospholipase C-beta2.

Signaling by guanine nucleotide-binding proteins (G proteins) involves sequential protein-protein interactions. G protein-betagamma subunit (Gbetagamma) interactions with phospholipase C-beta2 (PLC-beta2) were studied to determine if all Gbeta contacts are required for signaling. A peptide encoding Gbeta amino acid residues 86 to 105 stimulated PLC-beta2. Six residues (96 to 101) within this sequence could transfer signals and thus constitute a core signal transfer region. Another peptide, encoding Gbeta amino acid residues 115 to 135, did not substantially stimulate PLC-beta2 by itself but inhibited Gbetagamma stimulation, indicating that residues 115 to 135 constitute a general binding domain. Resolution of signal transfer regions from general binding domains indicates that all protein-protein contacts are not required for signal transfer and that it may be feasible to synthesize agonists and antagonists that regulate intracellular signal flow.

Occurrence of nocturia is not mediated by nocturnal hypoxia length and severity in patients with sleep-disordered breathing.

BACKGROUND: Nocturia is highly prevalent in subjects with respiratory sleep disturbances (ie obstructive sleep apnea). The aim of our study is to evaluate whether nocturia is associated with intermittent desaturations or hypoxia length and severity in people undergoing polysomnography. METHODS: We recruited 275 consecutive subjects attending the outpatient clinic for respiratory diseases at Campus Bio-Medico Teaching Hospital. Nocturia was defined as a self-reported voiding frequency ≥ two per night. The groups with and without nocturia were compared with parametric and non-parametric tests, as appropriated. Multivariable logistic regression analysis was used to assess the association of nocturia with patients' characteristics, including oxygen desaturation index (ODI), respiratory efforts (RE) and oxygen saturation below 90% (TST90). RESULTS: Sixty-six (24%) subjects reported nocturia, the median ODI was 15 (8-31), the median RE was 22 (12-38) and the median TST90 was 4.7 (0.3-20.6). ODI and RE were significantly higher in subjects with nocturia as compared with controls. In the multivariable model, ODI was associated with an increased probability of nocturia (OR = 1.03; 95% CI = 1.01-1.06), and the higher the ODI score, the higher the probability to have nocturia (P for trend = 0.038). No significant association was found between TST90 and the occurrence of nocturia. CONCLUSIONS: Intermittent desaturations and not hypoxia length and severity, expressed by TST90, are associated with the occurrence of nocturia in subjects complaining sleep disturbances.

[A correct nutritional approach as a primary goal in the management of chronic obstructive pulmonary disease]. / Una corretta modulazione nutrizionale come parte integrante del trattamento dei pazienti con BPCO.

The association between weight loss and COPD survival rate is known since the end of 19th century. Several studies showed how an insufficient caloric and nutritional uptake relates with higher mortality in COPD patients. Such clinical evidence brought a deep change in the management of this chronic condition, both in the hospital and home setting. Aim of this brief review is providing an update on the newest scientific evidences supporting the usefulness to systematically provide a complete nutritional evaluation to every COPD patient.

The effect of HIV-1 Gag myristoylation on membrane binding.

During the viral life cycle, an HIV protein, Gag, assembles at the host membrane, specifically at lipid raft regions, at very high concentrations leading to viral particle budding. Gag is post-translationally modified with an N-terminal myristate group which is thought to target Gag to lipid rafts thus aiding in assembly. Here we have analyzed the membrane binding of myristoylated HIV-1 Gag and a non-myristoylated form of HIV-1 Gag to various membrane models. After assessing the extent of myristoylation by HPLC and radiometric assays, we compared membrane binding using fluorescence methods. We found that myristoylated Gag shows a greater than twofold increase in binding affinity to model rafts. A structural model to explain these results is presented.

Developmental changes in the amount and functional state of UDP-glucuronosyltransferase.

The effect of postnatal development on the activity of liver microsomal UDP-glucuronosyltransferase was determined in male Wistar rats between 25 and 200 days of age using p-nitrophenol as aglycone. Enzyme activity (measured at 1.0 mM UDP-glucuronic acid, 0.05 mM p-nitrophenol) decreased 55% between 25 and 88 days of age and was constant thereafter. Treatment of microsomes with palmitoyl-lysophosphatidylcholine, which allows for an estimation of the amount of enzyme, showed approximately a four-fold decrease in enzyme concentration during the same period. This decrease was confirmed by Western blotting of microsomes with anti-UDP-glucuronosyltransferase antiserum. The fact that a nearly four-fold decline in enzyme concentration led to only a 55% decrease in activity indicates that there was an increase in activity per molecule of UDP-glucuronosyltransferase as the concentration of enzyme decreased. Treatment of microsomes with high pressure or detergent caused a greater extent of enzyme activation in microsomes prepared from 25 than 200 day old rats, suggesting that a fraction of the enzyme in older rats was activated in untreated microsomes. Fatty acid analysis of liver microsomal lipids during postnatal development revealed changes in docosahexaenoic acid (22:6) which correlated with levels of UDP-glucuronosyltransferase activity.

Thermodynamic characterization of the association of small basic peptides with membranes containing acidic lipids.

We measured the binding of the peptide acetyl-Trp-Lys7-amide to membranes formed from mixtures of the zwitterionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC) and the acidic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (PG). Direct filtration and equilibrium dialysis measurements demonstrate that binding increases sigmoidally with the mole fraction of PG as predicted from a simple Gouy-Chapman/mass action theoretical model. We used these measurements to calibrate two binding assays, one based on the increase in Trp fluorescence that occurs when the peptide binds to the membrane, the other on the quenching of Trp fluorescence that occurs when the peptide binds to membranes containing fluorescent lipids. Both fluorescence assays demonstrate that binding does not depend strongly on temperature, which suggests the enthalpy change, delta H, is small. Calorimetric measurements demonstrate this directly for the analogous basic peptide Lys5: delta H congruent to +1 kcal/mol for the binding of Lys5 to sonicated phospholipid vesicles and delta H congruent to 0 kcal/mol for its binding to large unilamellar vesicles. Thus, the decrease in the free energy that occurs when these peptides bind to the membrane is due to a positive change in the entropy of the system. Fluorescence measurements demonstrate the binding of the Trp-containing peptide to 4:1 PC/PG membranes is independent of pressure up to 2 kbar, which suggests that binding occurs without a significant change in volume.

The differential effects of carbon monoxide and oxygen on the pressure dissociation of Lumbricus terrestris hemoglobin.

We have explored the subunit affinities of Lumbricus terrestris hemoglobin (LtHb) under a variety of conditions using high-pressure spectroscopy. While only small changes were observed for LtHb-oxy below 1.0 kbar, higher pressures resulted in a 1000 cm-1 red shift and 2-fold increase in fluorescence intensity with a concomitant 12-fold decrease in scattering intensity, all of which reached completion by approx. 2.2 kbar. In the presence of 1 M MgCl2 or at acidic pH (4.2), the curves shifted by 400 and 1000 bar corresponding to significant destabilization. At pH 9.1, the initial spectral parameters were almost equal to the final endpoints and were unaffected by pressure. While the pressure curve of the CO form was similar to the oxy form at pH 7.2, the midpoints of the other samples were shifted to higher pressures relative to their oxy counterpart, indicating tighter subunit contacts. This stabilization was unexpected based upon the sequence homology to vertebrate hemoglobins, and the minimal structural differences between these two liganded forms of human hemoglobin. These data indicate that the differences are the result of the additive nature of the interactions involved in subunit packing whose effects become significant in larger aggregates.

Membrane-induced alterations in HIV-1 Gag and matrix protein-protein interactions.

The matrix (MA) domain of human immunodeficiency virus type 1 (HIV-1) contains sequences that direct association with the nucleus at early times in the virus replication cycle and with the plasma membrane at late times in the cycle. Localization to these sites is critical for functions related to the establishment of the infecting provirus and viral assembly, respectively. Mutational and structural analyses indicate that the opposing targeting signals which mediate these subcellular localization events include the same basic residues found in the N-terminal region of the protein. Here, we examined protein multimerization as a determinant of membrane association. Under high ionic strength conditions, Gag, but not MA, binds phospholipid membranes with high affinity. The oligomerization state of the protein per se did not appear to be a prerequisite for stable membrane binding, as Gag and MA were both capable of forming oligomers in high ionic strength buffer. To determine the fate of Gag and MA multimers in the presence of phospholipid membranes in real time, we measured resonance energy transfer between oligomer subunits in the presence and absence of lipid. The presence of phospholipid significantly increased the efficiency of resonance energy transfer between Gag molecules, consistent with enhanced Gag multimerization. This suggests that Gag oligomers assembled on the membrane surface and correlated with the observed stability of membrane binding. In contrast, the efficiency of resonance energy transfer between MA molecules decreased, indicating that MA oligomers dissociated in the presence of membrane, consistent with observed unstable binding. Identical results were obtained whether the probes were covalently attached to a Lys residue in Gag or to residues specifically within the MA domain of Gag; whether the fluorophore was rhodamine or fluorescein; or whether hetero- or homotransfer was measured. The results suggest that phospholipid induces alterations in Gag and MA protein-protein interactions that may contribute to the puzzling ability of MA to direct targeting functions requiring alternately membrane binding and membrane dissociation. The results also suggest that regions downstream of the MA domain in the precursor, or conformations formed after maturation of MA, play a critical role in oligomerization-modulated membrane binding.

Binding of equine infectious anemia virus matrix protein to membrane bilayers involves multiple interactions.

Human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) are closely related lentiviruses that infect immune cells, but their pathogenesis differ. Localization to the cytosolic leaflet of the plasma membrane is critical for replication of both viruses. This localization is accomplished through the matrix (MA) domain of the Gag precursor protein. In HIV-1, association of MA to anionic membranes appears to be primarily driven by a linear cluster of basic residues in the MA domain and an N-myristoylation signal. Interestingly, the MA protein of EIAV does not contain either of these signals. To understand which factors could promote EIAV assembly we characterized the membrane binding properties of its MA protein using fluorescence and biochemical methods. We find that EIAV MA exists as a multimer in solution whose protein-protein interactions are destabilized by membrane binding. EIAV MA binds strongly to electrically neutral membranes as well as to negatively charged membranes. Fluorescence quenching and chemical modification techniques, as well as trypsin proteolysis, indicate a different exposure of the EIAV MA Trp residues when bound to the two types of membranes, and EIAV MA proteolysis by trypsin differs when bound to the two types of membranes. Based on these data and the known structures of closely related matrix proteins, we constructed a structural model. This model predicts that EIAV MA binds to negatively charged membranes, but EIAV MA has an additional membrane binding region rich in residues that partition favorably into the membrane headgroup region. This secondary site may play a role in early events of viral infection.

The effect of hydrostatic pressure on membrane-bound proteins.

Many cellular proteins are bound to the surfaces of membranes and participate in various cell signaling responses. Interactions between this group of proteins are in part controlled by the membrane surface to which the proteins are bound. This review focuses on the effects of pressure on membrane-associated proteins. Initially, the effect of pressure on membrane surfaces and how pressure may perturb the membrane binding of proteins is discussed. Next, the effect of pressure on the activity and lateral association of proteins is considered. We then discuss how pressure can be used to gain insight into these types of proteins.

Endoscopic treatment of primary benign central airway tumors: Results from a large consecutive case series and decision making flow chart to address bronchoscopic excision.

BACKGROUND: Benign tracheo-bronchial neoplasms are rare, but potentially dangerous conditions with life threatening consequences. Tumor removal should be pursued by methods minimizing the procedural stress. The role of endoscopic treatment, as an alternative to open surgery, remains controversial. OBJECTIVES: report the twelve-years endoscopic experience in Rome, Italy. Fifty-seven benign tracheo-bronchial tumors were diagnosed and 130 tracheo-bronchial resections by rigid bronchoscopy performed. METHODS: we identified histotypes associated with higher recurrence rate and assessed their relationship with gender, age and tracheo-bronchial location. We provided data on safety and complications and suggested a decision making flow chart to address the patients to endoscopic resection. RESULTS: complete eradication after a single procedure without recurrence at 2 years was obtained in 63.1% of cases (36/57). Need of a second intervention within few months but no further recurrence at follow up was seen in a further 8.8% (5/57). Histotypes associated with recurrence were papillomas and inflammatory polyp. Seven patients (12.3%) were addressed to surgery because of multiple recurrence. Ten patients (17.5%) were lost at follow up. In case of recurrence, the bronchial biopsy was always repeated and no malignant transformation was observed. No major complications, pneumothorax or pneumomediastinum occurred. CONCLUSIONS: endoscopic treatment of benign tracheo bronchial tumors is safe and effective, provided that the procedure is carefully and systematically planned. The rate of eradication is satisfactory and the incidence of complications negligible. This will encourage this approach as first line treatment especially in patients, frequently elderly people, having increased surgical risk due to concomitant respiratory failure or major comorbidities.

Regulation of effectors by G-protein alpha- and beta gamma-subunits. Recent insights from studies of the phospholipase c-beta isoenzymes.

Both the alpha- and beta gamma-subunits of heterotrimeric guanine nucleotide-dependent regulatory proteins (G-proteins) couple members of the heptahelical class of cell-surface receptors to a diverse range of signal-generating effectors including retinal cyclic GMP phosphodiesterase, ion channels, adenylylcyclases, phosphoinositide 3-kinase, and members of the beta-class of inositol lipid-specific phospholipases C. Although the molecular details of the G-protein-regulated phospholipase C system were elucidated comparatively recently, these enzymes have become an important model for investigations of the process of G-protein effector coupling. A combination of molecular biological, biochemical, and structural studies using the phospholipase C-beta enzymes has provided some important insights into the interplay between G-proteins and their effectors and promises to reveal the mechanisms by which G-protein alpha- and beta gamma-subunits selectively associate with and activate effectors.

Binding properties of coumestrol to expressed human estrogen receptor.

We have studied the binding of coumestrol, an inherently fluorescent analog of 17 beta-estradiol, to human estrogen receptor (hER) in extracts of transfected cultured cells. The binding of coumestrol to the hER as well as its dissociation from the receptor can be directly determined by the change in fluorescence intensity of the probe. In agreement with previous studies using calf uterine extracts, we find that coumestrol binds to the receptor with a ten-fold lower affinity than 17 beta-estradiol. However, the rate of dissociation appears to be close to that of the native ligand. Coumestrol can accept energy from Trp residues in the excited state and, using a C530W hER mutant, we have confirmed that residue 530 is close to the ligand-binding pocket. We also find that the presence of saturating amounts of the specific DNA binding sites (ERE) did not significantly alter the binding affinity of coumestrol to either wild type hER or the C530W mutant.

Effects of cholesterol on membrane surfaces as studied by high-pressure fluorescence spectroscopy.

We have studied the effects of cholesterol on membrane surfaces using fluorescence spectroscopy at high pressure. At atmospheric pressure, the dissociation state of a pH-sensitive fluorophore (6-decanylnaphthol or DECNA) incorporated into several types of membranes showed an apparent increase in dissociation with cholesterol content coming somewhat closer to its dissociation state in solution. Previous studies have shown that when DECNA is free in solution, pressure induces proton dissociation due to the volume decrease that occurs when water electrostricts around the ions. But in phosphatidylcholine (PC) bilayers, proton dissociation is inhibited, either due to the inability of the surface to expand and allow for increased hydration, or other changes in lipid structure. The pressure behavior of DECNA in dioleoyl-PC, dioleoylphosphatidic acid and dioleylphosphatidylglycerol bilayers shows that incorporation of 5-10% cholesterol causes DECNA to behave like it is in a more unrestricted environment. This trend is reversed at higher cholesterol concentrations. These data, together with compressibility measurements, support the model of Sankaram and Thompson [M. Sankaram, T.E. Thompson, Biochemistry 29 (1990) 10676.] whereby in the disordered phase, cholesterol can span the two leaflets causing an increase in the area between the head groups; whereas in the ordered phase, no expansion occurs. Thus, the effect of cholesterol on membrane surfaces depends on its phase diagram.

Role of phosphatidylethanolamine lipids in the stabilization of protein-lipid contacts.

We have investigated the effect of lipids with phosphatidylethanolamine (PE) head groups on the stabilization of contacts between the tryptophan side chains of gramicidin and the lipid head groups. We initially developed two fluorescence methods that can be correlated to the spontaneous curvature of DOPC/DOPE and DOPC/DOPEme. One is based on bilayer structure and measures the rotational motion of a probe located close to the membrane surface relative to a more deeply-buried probe. The second is based on surface hydration/polarity and measures the emission energy of a polarity-sensitive probe located on the membrane surface. We used these methods to estimate the pseudo-curvature (i.e., curvature obtained by fluorescence measurements) of lipids with dimyristyl chains, and their pressure and temperature dependence. We then investigated the stability of gramicidin tryptophan-lipid contacts in DMPC/DMPE as a function of temperature and pressure. Stability was assessed by tryptophan rotational motion as determined by fluorescence anisotropy, since rotational motion is limited when the indoles are hydrogen bonded to the lipid head groups. The results suggest that the presence of PE lipids destabilizes these contacts due to either their smaller size relative to PC head groups, or their tendency to self-interact. Fluorescence quenching studies support these results.

The lateral fluidity of erythrocyte membranes. Temperature and pressure dependence.

The pressure and temperature dependence of the lateral and rotational fluidity of erythrocyte membranes was investigated by inserting the excimeric membrane probe 1'-pyrenedodecanoic acid (PDA) into the membranes of intact cells and measuring the probe excimer formation rate and the steady-state polarization of the monomer at pressures up to 2000 atm (2 kbar). At that pressure the lateral diffusivity of PDA was found to decrease by a factor of 10 and its emission anisotropy by a factor of 5 at 22 degrees C. At atmospheric pressure, the local lateral diffusion coefficient of PDA at 2 and 33 degrees C is 1.5 and 4.3 x 10(-8) cm2 s-1, respectively. The activation energy for probe translation was found to decrease from 6 to 3 kcal M-1 in going from atmospheric pressure to 2 kbar, while the entropy decreased by approx. 15 cal M-1 K-1, indicating greater lipid order at the high pressure. The experimental data are consistent with a 'free-area' model for the membrane, analogous to the free-volume model for nonassociated liquids. The lateral diffusivity of PDA was found to be proportional to the free membrane area and linear extrapolation to zero diffusivity indicates that at atmospheric pressure, the fractional free area of the erythrocyte membrane is 6%.